ABSTRACT This competitive renewal application is to investigate VPS35 (vacuolar protein sorting 35)?s function and mechanism in the pathogenesis of Alzheimer?s disease (AD). AD is the most common cause of dementia and the 6th leading cause of death in the USA. It is pathologically characterized by ?-amyloid (A?) deposits, neurofibrillary tangles, reactive glial-associated chronic inflammation, and pyramidal neuronal loss. Although A? is considered as a key mediator of the disease, recent studies suggest that the development of this complex disorder is regulated and contributed by multiple factors. VPS35-dysfunction is identified as one of the critical risk factors for AD. It is thus of considerable interest to investigate if and how VPS35-dysfunction promotes AD development. We have previously demonstrated that hemizygous deletion of the Vps35 gene in Tg2576 mice, a model of AD, precipitates A?-associated phenotypes [elevated A??levels, enhanced deficits in glutamatergic neurotransmission, long-term potentiation (LTP) in hippocampus, and memory impairment]. To explore underlying mechanisms, we first queried brain cell types that express VPS35. VPS35 is abundant in pyramidal neurons and microglia, both of which have been implicated in AD pathology. We then used knockdown (KD) and conditional knock out (cKO) approaches to investigate VPS35?s function. VPS35 KD or cKO in embryonic hippocampus led to axon spheroid formation, impairment in dendritic terminal growth, and decrease of spine density, phenotypes associated with neurodegenerative pathology. These phenotypes were development- specific, as KO VPS35 in embryonic (Vps35Nex), but not postnatal (Vps35Camk2a), pyramidal neurons resulted in these deficits. In Aim 1, we will address how VPS35-deficiency in developing pyramidal neurons causes neurodegenerative phenotypes. In addition to pyramidal neuronal VPS35, we investigated microglial VPS35?s function in AD pathology. Using a microglial selective Vps35 KO mouse line, Vps35CX3CR1, we found that microglial VPS35 is necessary to prevent hippocampal microglial activation and promote adult hippocampal neurogenesis, malfunction of both processes involved in AD pathogenesis. In Aim 2, we will further investigate if and how VPS35-loss in microglia contributes to AD pathogenesis. It is our hope that the results from this project will allow us to not only elucidate molecular and cellular mechanisms of VPS35 in AD development, but also open new avenues for therapeutic intervention to delay or prevent AD progression.